This project will determine how three closely related editosomes, precisely edit mRNAs and do so differentially between life cycle stages in Trypanosoma brucei. We hypothesize that insertion and deletion editosome compositional and structural differences enable differential binding and catalysis of specific gRNA/mRNA substrates during editing and the differential editing between developmental stages. We will: 1. Determine the high resolution structures of insertion and deletion editosomes, subcomplexes thereof, and RNA association by cryoEM. Samples for cryoEM will be purified from cells with one type of functional or catalytically arrested editosome. This will determine detailed editosomes architecture, protein stoichiometry, RNA location and differences between these editosomes. 2. Determine the roles non-catalytic editosome proteins/domains. We will determine if endonuclease partner proteins function as heterodimers and if noncatalytic proteins function in substrate RNA binding and positioning. The catalytic function of recombinant heterodimers will be assayed by crosslinking, mutagenesis and sequencing and functional RNA-protein interactions will be identified in vivo. 3. Determine how editosomes progress from one editing site (ES) to the next and test whether editing is either processive or progresses non-sequentially 3' to 5' and if endonuclease subcomplexes exchange between editosomes or not as they encounter different ESs. Cognate gRNA/mRNA pairs engaged in editing in cells with single or multiple types of functional editosomes will be identified and sequenced to resolve whether editing is processive or not. Proximal editosome specific proteins that will be tagged in vivo in cells that contain all three, combinations of two, one, or no functional editosomes that have specific tags and assayed to determine if editosome components exchange or not during editing. These results along RNAseq analysis of their edited RNAs will elucidate how the three different editosomes collaborate to edit multiple ESs specified by a single gRNA, including gRNAs that specify insertion and deletion. 4. Identify key aspects of developmental regulation of RNA editing. We will determine the order in which differentially edited mRNAs arise during development, if these are accompanied by differences in the abundances and editosome associations, of specific 3' initiating gRNAs and cognate gRNA/mRNA pairs, and if these are impacted by mutations that differentially affect editing. Selected structures of BF and PF editosomes will be compared by CXMS, SILAC and cryoEM based on Aim 1. These studies will determine whether specific cognate gRNA/mRNA pairs are differentially bound and utilized as a result of stage specific editosome differences. The project will provide key insights into how three poly- protein complexes function in an integrated fashion to precisely edit mt mRNAs and differentially regulate this process between life cycle stages which adapts energy metabolism to the different environments in the host and the vector.
This project will determine how different multi-protein catalytic complexes function together to change the sequences and thus meaning of multiple messenger RNAs that determine the composition of the cellular energy generating machinery in an important parasite and do so differentially during its development stages while adapting it to the human host and the insect that transmits it. It will provide insights into how various protein/RNA complexes which are present in all higher organisms, including humans, function and how such information may be used for drug development.
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